GTF2F1 Antibody

What is GTF2F1 and what is its functional role in transcription?

GTF2F1, also known as RAP74, BTF4, TF2F1, or TFIIF, is the 74 kDa subunit of general transcription factor IIF. It plays a crucial role in eukaryotic gene transcription, specifically in the initiation of transcription by RNA polymerase II. The protein enables several functions, including RNA polymerase II general transcription initiation factor activity, phosphatase activator activity, and promoter-specific chromatin binding activity . GTF2F1 is involved in positive regulation of transcription by RNA polymerase II, response to virus, and is a component of both the transcription factor TFIID complex and transcription factor TFIIF complex . In the cellular context, GTF2F1 is primarily located in cell junctions and the nucleoplasm, where it participates in the ordered assembly of multiprotein complexes on proximal promoter elements .

What applications are GTF2F1 antibodies routinely used for?

GTF2F1 antibodies are validated for multiple experimental applications in molecular and cellular biology research:

The antibodies have been successfully employed in multiple studies focusing on transcription regulation mechanisms and have shown consistent performance across these applications . For optimal results, it is recommended that researchers titrate the antibody in each testing system to determine ideal conditions for their specific experimental setup .

What species reactivity do commercially available GTF2F1 antibodies demonstrate?

Most commercial GTF2F1 antibodies show cross-reactivity with multiple mammalian species. Based on the validated data:

The species reactivity is particularly important when designing experiments across different model systems . When using these antibodies in species not explicitly listed in the tested reactivity, additional validation steps should be performed to confirm specificity and performance.

How do researchers address the molecular weight discrepancy between calculated and observed GTF2F1 values?

A notable observation with GTF2F1 is the discrepancy between its calculated molecular weight (58 kDa) and observed molecular weight (74 kDa) in SDS-PAGE gels . This difference is likely due to post-translational modifications or structural characteristics affecting protein migration.

When analyzing western blot results for GTF2F1, researchers should expect to observe bands at approximately 74 kDa rather than at the calculated 58 kDa position . This discrepancy should be accounted for when interpreting results and can be addressed by:

• Running appropriate positive controls with known GTF2F1 expression (such as K-562 cell lysates)

• Including molecular weight markers covering the 50-100 kDa range

• Performing validation with multiple GTF2F1 antibodies recognizing different epitopes

• Considering phosphorylation states that may affect electrophoretic mobility

Understanding this migration pattern is crucial for accurate identification of GTF2F1 in experimental samples and avoiding misinterpretation of western blot results .

What are the optimal conditions for using GTF2F1 antibodies in immunohistochemistry applications?

For successful immunohistochemical detection of GTF2F1, several critical parameters must be optimized:

Antigen retrieval is particularly important for GTF2F1 detection in fixed tissues. The recommended approach uses TE buffer at pH 9.0, though citrate buffer at pH 6.0 provides an alternative method if TE buffer yields suboptimal results . The specific choice between these methods may depend on tissue type, fixation duration, and protocol-specific factors.

For highly reproducible results, researchers should conduct preliminary optimization experiments with positive control tissues (such as mouse lung or rat ovary) where GTF2F1 expression has been confirmed . This optimization process should include testing multiple dilutions to determine the ideal antibody concentration that maximizes specific signal while minimizing background staining.

What controls and validation approaches are essential when using GTF2F1 antibodies?

Rigorous validation is crucial for ensuring reliable results with GTF2F1 antibodies. Comprehensive validation should include:

• Positive Controls: Include tissues or cell lines with known GTF2F1 expression (K-562 cells have been validated for western blot and immunoprecipitation applications)

• Negative Controls:

  • Primary antibody omission

  • Isotype controls (rabbit IgG for polyclonal antibodies)

  • Tissues/cells with confirmed absence or knockdown of GTF2F1

• Antibody Specificity Testing:

  • Pre-adsorption with immunizing peptide

  • Comparison with multiple antibodies targeting different GTF2F1 epitopes

  • Genetic validation using CRISPR knockout or siRNA knockdown samples

• Cross-Reactivity Assessment:

  • Testing multiple species if cross-species applications are planned

  • Western blot confirmation prior to immunohistochemistry or immunoprecipitation

These validation steps are particularly important when studying GTF2F1 in new experimental contexts or when using the antibody in species not previously tested .

What are the technical considerations for RNA immunoprecipitation studies with GTF2F1 antibodies?

RNA immunoprecipitation (RIP) has emerged as an important application for GTF2F1 antibodies, as indicated by published research utilizing GeneTex GTF2F1 antibody (GTX114455) at 0.5 μg/ml concentration . When performing RIP with GTF2F1 antibodies, researchers should consider:

• Crosslinking Conditions:

  • Formaldehyde crosslinking (1-3%) for protein-RNA interactions

  • UV crosslinking as an alternative approach for direct protein-RNA contacts

• Lysis Buffer Composition:

  • Include RNase inhibitors to prevent RNA degradation

  • Consider detergent concentrations that maintain protein-RNA complexes

  • Add protease inhibitors to prevent protein degradation

• Antibody Selection and Amount:

  • Use antibodies validated specifically for RIP (such as GeneTex GTX114455)

  • Titrate antibody amounts (typically starting with 0.5-5 μg per reaction)

  • Confirm specificity via western blot prior to RIP experiments

• Controls:

  • Include IgG isotype control IP

  • Perform RT-PCR for housekeeping genes/non-targets

  • Include input samples representing pre-IP material

• Downstream Analysis:

  • RT-PCR for candidate RNA targets

  • RNA-seq for unbiased identification of bound RNAs

  • qPCR validation of identified targets

This application is particularly valuable for investigating GTF2F1's potential roles in RNA binding and post-transcriptional regulation beyond its canonical role in transcription initiation .

How should researchers interpret GTF2F1 antibody results in the context of transcription complex studies?

GTF2F1 functions as part of multiprotein transcription complexes, specifically the transcription factor TFIID complex and TFIIF complex . When designing experiments to study these complexes:

• Co-immunoprecipitation Approaches:

  • GTF2F1 antibodies can be used to pull down associated proteins

  • Careful buffer optimization is needed to maintain complex integrity

  • Combination with mass spectrometry can identify novel interaction partners

• Chromatin Immunoprecipitation (ChIP):

  • GTF2F1 antibodies can identify genomic binding sites

  • Results should be interpreted in relation to RNA Polymerase II binding

  • Expected enrichment at promoter regions of actively transcribed genes

• Proximity Ligation Assays:

  • Can detect GTF2F1 interactions with other transcription factors in situ

  • Provides spatial information about complex formation in nuclei

• Dual Immunofluorescence:

  • Co-localization studies with other transcription machinery components

  • Nuclear localization patterns can indicate transcriptional activity states

When interpreting results, researchers should consider GTF2F1's dual residence in transcription factor TFIID and TFIIF complexes, as this may reflect different functional states or regulatory mechanisms . Additionally, the detection of GTF2F1 at specific genomic loci or in specific nuclear compartments can provide insights into the dynamics of transcription initiation.

What methodological approaches are recommended for studying GTF2F1 in virus response mechanisms?

GTF2F1 has been implicated in cellular responses to viral infection . Researchers investigating this aspect should consider:

• Infection Models:

  • Select appropriate viral systems based on research question

  • Include time course studies to capture dynamic responses

  • Consider both lytic and latent infection models

• GTF2F1 Expression Analysis:

  • Western blot with GTF2F1 antibodies to assess protein level changes

  • Quantitative comparison between infected and uninfected samples

  • Subcellular fractionation to detect relocalization events

• Functional Studies:

  • ChIP-seq to identify changes in GTF2F1 genome occupancy following infection

  • RNA-seq combined with GTF2F1 knockdown to identify regulated genes

  • Co-immunoprecipitation to detect virus-induced changes in protein interactions

• Data Interpretation Frameworks:

  • Compare GTF2F1 behavior with other general transcription factors

  • Correlate GTF2F1 changes with viral gene expression phases

  • Assess relationship to interferon response gene activation

This research direction may provide insights into how viruses manipulate host transcription machinery and how general transcription factors like GTF2F1 contribute to antiviral responses .

What approaches are recommended for troubleshooting non-specific binding with GTF2F1 antibodies?

Non-specific binding is a common challenge when working with antibodies against transcription factors like GTF2F1. When encountering this issue:

• Antibody Dilution Optimization:

  • Test multiple dilutions within and beyond the recommended range (1:500-1:2000 for WB/IHC)

  • Create a dilution series to identify optimal signal-to-noise ratio

  • Document results systematically for different applications

• Blocking Conditions:

  • Test alternative blocking agents (BSA, milk, commercial blockers)

  • Optimize blocking time and temperature

  • Consider adding 0.1-0.5% Tween-20 to reduce non-specific hydrophobic interactions

• Washing Stringency:

  • Increase number of washes

  • Adjust salt concentration in wash buffers

  • Consider adding detergents to wash buffers

• Antibody Cross-Adsorption:

  • Pre-incubate with tissues/cells lacking GTF2F1 expression

  • Consider commercial cross-adsorption services for problematic antibodies

• Sample Preparation:

  • Optimize protein extraction methods

  • Consider nuclear extraction protocols for enrichment

  • Test different fixation methods for IHC applications

By systematically addressing these parameters, researchers can typically reduce non-specific binding while maintaining specific GTF2F1 detection .

How can GTF2F1 antibodies be utilized in ELISA-based quantification systems?

ELISA kits for GTF2F1 detection, such as the Assay Genie Mouse GTF2F1 ELISA Kit, employ sandwich assay principles for specific quantification. When utilizing such systems:

• Assay Characteristics:

  • Sensitivity: 0.056 ng/mL

  • Detection range: 0.16-10 ng/mL

  • Standard concentration: 10 ng/mL

  • Assay duration: 3.5 hours

• Sample Types Compatible:

  • Tissue homogenates

  • Cell lysates

  • Other biological fluids

• Methodological Principles:

  • Pre-coated microplate with GTF2F1-specific antibody

  • Addition of samples or standards

  • Addition of biotin-conjugated GTF2F1-specific antibody

  • Addition of Avidin-HRP conjugate

  • TMB substrate reaction

  • Absorbance measurement at 450nm ± 10nm

• Performance Metrics:

  • Intra-assay precision: CV% < 8%

  • Inter-assay precision: CV% < 10%

Researchers should prepare samples according to kit-specific instructions and consider performing pilot studies to determine optimal sample dilutions. The resulting quantitative data can be particularly valuable for comparative studies examining GTF2F1 expression levels across different experimental conditions or tissue types .

What methodological considerations apply to studying GTF2F1 genetic variants?

The GTF2F1 gene (ID: 2962) is located on chromosome 19p13.3 and comprises 14 exons. When investigating GTF2F1 genetic variants:

• Genomic Context:

  • Chromosome location: 19p13.3

  • Genomic coordinates: NC_000019.10 (6379572..6393164, complement)

  • Total exons: 14

• Variant Analysis Resources:

  • ClinVar for reported clinical variants

  • dbVar for structural variants

  • SNP database for single nucleotide polymorphisms

  • NCBI Variation Viewer for comprehensive variant visualization

• Experimental Approaches:

  • Targeted sequencing of GTF2F1 coding regions

  • Functional studies of variants using reporter assays

  • CRISPR-based modeling of specific variants

  • Protein structure analysis of variant effects

• Data Interpretation:

  • Assess evolutionary conservation at variant positions

  • Predict functional impact using in silico tools

  • Correlate with known transcriptional defects

  • Consider impact on protein-protein interactions

Understanding genetic variation in GTF2F1 can provide insights into potential altered functionality in transcription initiation and may have implications for disease mechanisms related to transcriptional dysregulation .

What emerging applications for GTF2F1 antibodies show promise for future research?

Several cutting-edge approaches offer new opportunities for GTF2F1 research:

• Single-Cell Applications:

  • Mass cytometry with GTF2F1 antibodies for single-cell protein measurement

  • Integration with single-cell transcriptomics for multi-omic analysis

  • Spatial transcriptomics correlated with GTF2F1 immunohistochemistry

• Live-Cell Imaging:

  • Development of non-disruptive labeling techniques for GTF2F1

  • Real-time visualization of transcription complex assembly

  • FRAP (Fluorescence Recovery After Photobleaching) for dynamics studies

• Structural Biology Integration:

  • Combining antibody-based techniques with cryo-EM studies

  • Validating structural models with epitope-specific antibodies

  • Conformational state-specific antibodies for functional studies

• Therapeutic Applications:

  • GTF2F1 dysregulation in disease contexts

  • Antibody-based detection of altered GTF2F1 as biomarkers

  • Targeted disruption of specific GTF2F1 interactions

These emerging applications will benefit from continued refinement of antibody specificity and validation protocols. As methodologies advance, researchers will gain increasingly detailed insights into the dynamics and regulatory mechanisms of GTF2F1 in transcription initiation and beyond.

How should researchers integrate multiple detection methods for comprehensive GTF2F1 analysis?

A multi-method approach provides the most complete picture of GTF2F1 biology:

• Complementary Detection Strategies:

  • Western blot for protein expression levels and modifications

  • IHC for spatial distribution in tissues

  • IP for interacting partners

  • ELISA for quantitative measurement

  • ChIP for genomic binding sites

• Integration Frameworks:

  • Correlate protein expression (WB/ELISA) with genomic occupancy (ChIP)

  • Connect tissue localization (IHC) with interaction networks (IP-MS)

  • Link expression changes to functional outcomes through knockdown studies

• Data Integration Approaches:

  • Consistent sample processing across methods

  • Parallel analysis of the same experimental conditions

  • Computational integration of multi-omic datasets

  • Validation across different antibodies and detection methods